1. Field of Invention
The present invention is directed to genes encoding plant fatty acid synthase enzymes relevant to fatty acid synthesis in plants, and to methods of using such genes in combination with genes encoding plant medium-chain preferring thioesterase proteins. Such uses provide a method to increase the levels of medium-chain fatty acids that may be produced in seed oils of transgenic plants.
2. Background
Higher plants synthesize fatty acids via a common metabolic pathway. In developing seeds, where fatty acids attached to triglycerides are stored as a source of energy for further germination, the fatty acid synthesis pathway is located in the plastids. The first step is the formation of acetyl-ACP (acyl carrier protein) from acetyl-CoA and ACP catalyzed by a short chain preferring condensing enzyme, xcex2-ketoacyl-ACP synthase (KAS) III. Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves the cyclical action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP to form a longer xcex2-ketoacyl-ACP (xcex2-ketoacyl-ACP synthase), reduction of the keto-function to an alcohol (xcex2-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (xcex2-hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACP reductase). xcex2-ketoacyl-ACP synthase I (KAS I), is primarily responsible for elongation up to palmitoyl-ACP (C16:0), whereas xcex2-ketoacyl-ACP synthase II (KAS II) is predominantly responsible for the final elongation to stearoyl-ACP (C18:0).
Genes encoding peptide components of xcex2-ketoacyl-ACP synthases I and II have been cloned from a number of higher plant species, including castor (Ricinus communis) and Brassica species (U.S. Pat. No. 5,510,255). KAS I activity was associated with a single synthase protein factor having an approximate molecular weight of 50 kD (synthase factor B) and KAS II activity was associated with a combination of two synthase protein factors, the 50 kD synthase factor B and a 46 kd protein designated synthase factor A. Cloning and sequence of a plant gene encoding a KAS III protein has been reported by Tai and Jaworski (Plant Physiol. (1993) 103:1361-1367).
The end products of plant fatty acid synthetase activities are usually 16- and 18-carbon fatty acids. There are, however, several plant families that store large amounts of 8- to 14-carbon (medium-chain) fatty acids in their oilseeds. Recent studies with Umbellularia californica (California bay), a plant that produces seed oil rich in lauric acid (12:0), have demonstrated the existence of a medium-chain-specific isozyme of acyl-ACP thioesterase in the seed plastids. Subsequent purification of the 12:0-ACP thioesterase from Umbellularia californica led to the cloning of a thioesterase cDNA which was expressed in seeds of Arabidopsis and Brassica resulting in a substantial accumulation of lauric acid in the triglyceride pools of these transgenic seeds (U.S. Pat. No. 5,512,482). These results and subsequent studies with medium-chain thioesterases from other plant species have confirmed the chain-length-determining role of acyl-ACP thioesterases during de novo fatty acid biosynthesis (T. Voelker (1996) Genetic Engineering, Ed. J. K. Setlow, Vol. 18, pgs. 111-133).